Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4α and HNF1β in a pancreatic β-cell line -: art. no. e150
Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4α and HNF1β in a pancreatic β-cell line -: art. no. e150
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DOI：
10.1093/nar/gnh144
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Ryffel, GU
中科院分区：
文献类型：
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作者：
Thomas, H;Senkel, S;Ryffel, GU
使用大鼠胰岛素瘤细胞系INS-1，我们产生了对基因转移最有效的β细胞克隆，因为它们含有Flp重组酶介导的单拷贝转基因定点整合的FRT位点。因此，可以通过DNA转染引入感兴趣的基因，而不需要选择单个细胞克隆。此外，克隆含有四环素阻遏物，允许四环素诱导转基因。通过寡核苷酸芯片，我们确定了Flp-In T-REx细胞克隆的β细胞特异性表型。利用有限水平表达HNF 6、HNF 4 α和HNF 1 β转录因子的克隆，我们引入了编码这些因子的表达载体。我们通过蛋白质印迹和免疫细胞化学显示了四环素对这些转录因子的有效诱导。微阵列显示，这三个因素影响相似数量的基因，只有少数基因共同调控。统计分析表明，这三个转录因子影响的基因分类到不同的生物过程。此外，我们还记录了这些Flp-In T-REx细胞对人类MODY 5患者中发现的突变HNF 1 β转录因子的功能分析的有用性。我们发现突变体P328 L329 del和A263 insGG的表达只影响很少的转录本，这些转录本主要与野生型HNF 1 β诱导的转录本不同。
Using the rat insulinoma cell line INS-1 we generated beta-cell clones that are most efficient for gene transfer, as they contain an FRT site for Flp recombinase-mediated, site-directed integration of a single copy transgene. Therefore, the gene-of-interest can be introduced by DNA transfection without the need to select individual cell clones. Additionally, the clones contain the tetracycline repressor allowing tetracycline induction of the transgene. By oligonucleotide microarray we define the beta-cell specific phenotype of the Flp-In T-REx cell clones. Using a clone expressing the HNF6, HNF4alpha and HNF1beta transcription factors at a limited level, we introduced the expression vectors encoding these factors. We show efficient tetracycline induction of these transcription factors by western blots and immunocytochemistry. Microarrays reveal that these three factors affect a similar number of genes with only few genes regulated in common. Statistical analysis reveals that the three transcription factors affect genes categorized to different biological processes. Furthermore, we document the usefulness of these Flp-In T-REx cells for the functional analysis of mutated HNF1beta transcription factors found in human MODY5 patients. We show that the expression of the mutant P328L329del and A263insGG affects only very few transcripts and these are predominantly distinct from those induced by wild-type HNF1beta.